[0001] The present invention is directed to the use of allogenic effector cells together
with trifunctional bispecific or trispecific antibodies or a combination thereof in
order to substantially reduce or avoid a graft versus host disease in the treatment
of tumor growth and metastasis in a mammal.
[0002] Therapeutic options for patients with recurrent leukemia or B cell lymphoma refractory
to the standard therapy are limited. A promising approach for such resistant malignancies
is immunotherapy with e.g. donor lymphocyte infusions (1-3). Donor lymphocyte infusions
can cure especially recurrent chronic myelogenous leukemia, probably by the action
of T cells, but is complicated by the possibility of simultaneously induced graft-versus-host
disease. Therefore, a more specific and better controllable therapy, e.g. with monoclonal
antibodies that target lymphocytes to residual tumor cells, is desired. However, in
most cases, unlabeled or unconjugated antibodies do not activate immune effector mechanisms
sufficiently to eradicate all tumor cells. Thus, T cell redirecting bispecific antibodies
(bsAb), which combine the cell-specificity of monospecific antibodies with the potency
of T cells may be more effective.
[0003] Bispecific antibodies consisting of 2 different antigenic specificities have been
developed as immunotherapeutic reagents for targeting immune cells to tumor tissue
(4-8). This strategy is based on the assumption that appropriate effector/target cell
interaction via a physical contact between immune cells and tumor cells, activates
cytotoxic mechanisms that lead to an efficient eradication of tumor cells.
[0004] Many different bispecific antibody formats have been created over the last 20 years
with varying qualities in production,
in vitro and in vivo efficacy, recruitment of effector cells or e.g. need for additional T cell stimuli
(9-13). Finally, several of these constructs reached the clinic with minor to moderate
and only sometimes promising efficacy (14-17). Trifunctional antibodies (trAb) are
artificially engineered immunoglobulins with an unique composition of heavy chains
of mouse IgG2a and rat IgG2b, representing highly homologous Ig subclasses. Both isotypes
are very potent in terms of immunological effector functions, such as complement dependent
cytotoxicity (CDC) and antibody dependent cell-mediated cytotoxicity (ADCC). Noteworthy,
the Fc region composed by these two subclasses, effectively binds to human FcγI and
III-receptors on accessory cells (like e.g. macrophages, dendritic cells and natural
killer cells) but not to the inhibitory Fcγ receptor type II expressed e.g. on B cells.
As a consequence trAbs can not only redirect T-cells to tumor cells, but also induce
recruitment and activation of accessory cells through their Fc region. The simultaneous
activation of different mechanisms at the tumor site such as phagocytosis, perforin
mediated lysis and cytokine release results in a particularly efficient destruction
of tumor cells (18, 19). Remarkably, also apoptosis-resistant tumor cells can be eliminated
by this process (19). As a further consequence of the so induced uptake of tumor material
by the antigen presenting system even a long-lasting protective antitumor immunity
could be established as already demonstrated in two immunocompetent murine tumor models
(20).
[0005] While most BsAb treatments aiming to achieve anti-tumor response have been combined
with syngeneic/autologous derived cells, we and others (3, 10, 21-23) have also used
an immunotherapeutic strategy based on the allogeneic reaction of major histocompatibly
mismatched cells, known in clinical practice as donor lymphocyte infusion (DLI), following
hematopoietic stem cell transplantation (SCT). Unfortunately, the use of allogeneic
cell therapy (alloCT) in the context of currently applicable protocols in experimental
models and in clinical practice, is frequently accompanied by life-threatening acute
and chronic graft versus host disease (GVHD) that is difficult to control effectively
with currently available treatments (23-28).
[0006] It is therefore an object of the present invention to provide a new therapeutic method
for treating tumor growth and metastasis in a mammal, preferably a human, wherein
life-threatening acute and chronic graft versus host disease (GVHD) is either efficiently
reduced to a level not threatening the patient's life or is even avoided. It is a
further object of the present invention to provide the use of allogeneic effector
cells together with trifunctional bispecific or trispecific antibodies for treating
tumor growth and metastasis in a mammal.
[0007] This object is achieved by providing the use of allogeneic effector cells together
with trifunctional bispecific or trispecific antibodies having the following properties:
a) binding to a T cell
b) binding to at least one antigen on a tumor cell
c) binding via their Fc portion in the case of trifunctional bispecific antibodies
or via a third specificity in the case of trispecific antibodies to Fc receptor positive
cells,
said antibodies redirecting the allogeneic cells away from host tissues in order to
substantially reduce or avoid a graft versus host disease for the manufacture of a
medicament for treating tumor growth and metastasis in a mammal, preferably a human.
[0008] It is a further object of the present invention to provide a new therapeutic composition
for treating tumor growth and metastasis in a mammal, preferably a human, wherein
life-threatening acute and chronic graft versus host disease (GVHD) is either efficiently
reduced to a level not threatening the patient's life or is even avoided.
[0009] This object is achieved by providing a pharmaceutical composition for treating tumor
growth and metastasis in a mammal, said composition comprising a therapeutically effective
amount of allogeneic effector cells together with trifunctional antibodies having
the following properties:
a) binding to a T cell
b) binding to at least one antigen on a tumor cell
c) binding via their Fc portion in the case of trifunctional bispecific antibodies
or via a third specificity in the case of trispecific antibodies to Fc receptor positive
cells together with pharmaceutically acceptable carriers and auxiliary substances.
[0010] Preferred embodiments are further described in the following description, the experimental
section in combination with the figures and the enclosed claims.
[0011] It has surprisingly been found that a combination of trifunctional bispecific antibodies
(trAbs) and/or trispecific antibodies, as defined herein, with allogeneic effector
cells (said method called allogeneic cell therapy = alloCT) allows to direct alloreactive
effector cells to tumor tissue rather than to normal host cells, thereby allowing
development of more efficient graft-versus-tumor (GVT) effect as well as to minimize
the risk of GVHD. In the here presented rationale, a therapy is designed to treat
recipients with cancer cells, expressing tumor-associated antigens (e.g. EpCAM, Her2/neu
or CD20) as the targeting molecule, with trifunctional bispecific antibodies and/or
trispecific antibodies, given together with allogeneic effector cells, particularly
donor lymphocytes and/or myeloid cells, in order to assess their capability to selectively
eliminate tumor cells while sparing normal host cells in an attempt to avoid lethal
GVHD.
BRIEF DESCRIPTION OF THE FIGURES
[0012] The invention will be illustrated in more detail by means of the accompanying Figures.
The Figures show:
Figure 1. The effect of allogeneic cell therapy with or without BiLu on development
of GVHD assessed by measurement of body weight.
[0013] BALB/cXC57BL/6)F
1 mice were inoculated intraperitoneally (IP) with 5X10
4 B16-EpCAM melanoma cells 24h after conditioning with TBI 4Gy. One day later, 30X10
6 naive or rIL-2 activated C57BL/6 splenocytes were administered IP with or without
pretreatment with BiLu antibodies (10µg/mouse).
Figure 2. The role of tumor burden on susceptibility to GVHD induced by alloreactive
spleen cells with or without pretreatment with BiLu.
[0014] Sublethally irradiated (BALB/cXC57BL/6)F
1 mice were conditioned with TBI 4Gy. One day following TBI, mice were injected IP
with 5X10
3 or 5X10
4 B16-EpCAM melanoma cells or left without tumor inoculation. One day later, mice were
inoculated intraperitoneally (IP) with 30X10
6 naïve C57BL/6 splenocytes with or without pretreatment with BiLu (10µg/mouse) in
order to determine the possible role of tumor existence on targeting of alloreactive
lymphocytes, guided by BiLu, to the tumor for desirable prevention of GVHD while attempting
to maximize GVT effects.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0015] The trifunctional antibodies to be used in the present invention are known in the
art and are described in several documents. Reference is made e.g. to US 6,551,592,
US2003223999, US2002051780, and US6210668 and the articles indicated above; all of
these documents and the following references are fully incorporated by reference into
this application.
[0016] The antibodies to be used in the present invention are preferably characterized by
the additional effects of
- activation of the Fc receptor-positive cell by binding to the Fc receptor-positive
cell via Fcγ receptors of type I and III (CD64 and CD16) (18) and, thereby, initiating
or increasing the expression of cytokines and/or costimulatory antigens; and
- transfer of at least a second activation signal, required for physiological activation
of the T cell, to the T cell by the co-stimulatory antigens and/or cytokines, this
activation being indicated by up-regulation of activation markers, killing of the
tumor cell, and/or T cell proliferation.
[0017] Preferably, the antibodies used in the method and composition of the present invention
are also able to activate tumor-specific T cells recognizing a tumor-specific peptide
presented on the tumor cells by MHC class I and/or class II via their T cell receptor
upon binding to the trifunctional bispecific or trispecific antibody as described
herein.
[0018] Further, the antibodies used according to the invention are preferably able to reactivate
the tumor-specific T cells being in an anergic state. Furthermore, they are preferably
able to induce tumor-reactive complement-binding antibodies and, thus, induce a humoral
immune reaction.
[0019] Binding to the T cell takes place via CD3, CD2, CD5, CD28, and/or CD44. The Fc receptor-positive
cells have at least one Fcγ receptor I or III.
[0020] The antibody used according to the invention is able to bind to monocytes, macrophages,
and/or dendritic cells being Fcγ receptor I-positive cells.
[0021] The antibodies used according to the invention lead to the initiation or increase
of the expression of CD40, CD80, CD86, ICAM-1, and/or LFA-3 being co-stimulatory antigens
and/or secretion of cytokines by the Fc receptor-positive cell. Preferably, the cytokines
are IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-12, INF-gamma and/or TNF-[alpha].
[0022] Preferably, binding to the T cell takes place via the T cell receptor complex of
the T-cell.
[0023] The trifunctional bispecific antibody used in the invention preferably is an anti-CD3
X anti-tumor-associated antigen antibody and/or anti-CD2 X anti-tumor-associated antigen
antibody and/or anti-CD5 X anti-tumor-associated antigen antibody and/or anti-CD28
X anti-tumor-associated antigen antibody and/or anti-CD44 X anti-tumor-associated
antigen antibody.
[0024] The trispecific antibody used according to the invention preferably is an anti-CD3
X anti-tumor-associated antigen antibody and/or anti-CD2 X anti-tumor-associated antigen
antibody and/or anti-CD5 X anti-tumor-associated antigen antibody and/or anti-CD28
X anti-tumor-associated antigen antibody and/or anti-CD44 X anti-tumor-associated
antigen antibody having an additional anti-Fc receptor binding arm.
[0025] Regarding feature (a), the first signal is for example transduced via the T cell
receptor complex of the T cell and, therefore, may itself lead to an unphysiological
T cell activation. By this, the cell could be anergized and unable to react to T cell
receptor-mediated stimuli. In addition, a second activation signal is transduced to
the T cell by the trifunctional bispecific or trispecific antibodies of the invention
via the co-stimulatory antigens on the simultaneously bound Fc receptor-positive cell
which causes physiological activation of the T cell and, subsequently, leads to killing
of the tumor cell and/or proliferation of the T cell. As a further criterion for T
cell activation the up-regulation of cell surface antigens such as CD2, CD25, and/or
CD28, and/or the secretion of cytokines such as e.g. IL-2 or INF-gamma may be used.
[0026] Thus, by the use of the trAbs described according to the invention T cells and accessory
cells are activated and retargeted simultaneously against the tumor cells.
[0027] Preferred antibodies are heterologous trifunctional bispecific antibodies selected
of one or more of the following combinations of isotypes:
- rat-IgG2b/mouse-IgG2a,
- rat-IgG2b/mouse-IgG2b,
- rat-IgG2b/mouse-IgG3;
- rat-IgG2b/human-IgG1,
- rat-IgG2b/human-IgG2
- rat-IgG2b/human-IgG3 [oriental allotype G3m(st)=binding to protein A], rat-IgG2b/human-IgG4;
- rat-IgG2b/rat-IgG2c;
- mouse-IgG2a/human-IgG3 [caucasian allotypes G3m(b+g)=no binding to protein A, in the
following indicated as *]
- mouse-IgG2a/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- mouse-IgG2a/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge] -human-IgG3*-[CH2-CH3]
- mouse-IgG2a/human-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- mouse-[VH-CH1, VL-CL]-human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- mouse-[VH-CH1, VL-CL]-human-IgG4/rat-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG4
[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
- at-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge-CH2-CH3]
- rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG2-[hinge-CH2-CH3]
- rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG3-[hinge-CH2-CH3, oriental allotype]
- rat-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG4-[hinge-CH2-CH3]
- human-IgG1/human-[VH-CH1, VL-CL]-human-IgG1-[hinge] -human-IgG3*-[CH2-CH3]
- human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG4 [N-terminal region of
CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
- human-IgG1/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG4 [N-terminal region
of CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
- human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG2 [N-terminal region of
CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
human-IgG1/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG2 [N-terminal region
of CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
- human-IgG1/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- human-IgG1/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*- [CH2-CH3]
- human-IgG2/human-[VH-CH1, VL-CL]-human-IgG2-[hinge]-human-IgG3*-[CH2-CH3]
- human-IgG4/human-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG3 *-[CH2-CH3]
- human-IgG4/human-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG4 [N-terminal region
of CH2]-human-IgG3*[C-terminal region of CH2:>aa position 251]-human-IgG3*[CH3]
- mouse-IgG2b/rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- mouse-IgG2b/human-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
- mouse-IgG2b/mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]
[0028] The trifunctional bispecific and trispecific antibodies described in the use and
method of the present invention are characterized in particular by the features described
in the claims and preferably of course by the features (a)-(c) described in claim
1. Thus, these antibodies may be used together with allogeneic effector cells in humans
and animals in a pharmaceutical preparation, e.g. in donor lymphocyte infusions.
[0029] Preferably, specific subclasses or combinations of subclasses, respectively, of the
trAb or in the case of trispecific antibodies a binding arm recognizing the Fc receptor
are employed for the activation of the Fc receptor-positive cell. For
example, in vitro experiments showed that intact bsAb of the mouse-IgG2a/rat-IgG2b subclass combination
are able to bind to and simultaneously activate Fc receptor-positive cells leading
to up-regulation or new formation (expression), respectively, of co-stimulatory antigens
such as e.g. CD40, CD80, or CD86 on the surface of these cells, whereas bsAb of the
mouse-IgG1/rat-IgG2b subclass combination are able to bind to Fc receptor-positive
cells (Haagen et al., Interaction of human monocyte Fc[gamma] receptors with rat IgG2b,
J. Immunology, 1995, 154:1852-1860) but obviously are unable to activate these cells
to a comparable extent (Gast G. C., Haagen I. -A., van Houten A. A., Klein S., Duits
A. J., de Weger R. A., Vroom T. M., Clark M. R., J. Phillips, van Dijk A. J. G., de
Lau W. B. M., Bast B. J. E. G. CD8 T-cell activation after intravenous administration
of CD3*CD19 bispecific antibody in patients with non-Hodgkin lymphoma. Cancer Immunol.
Immunother. 40:390, 1995).
[0030] While the intact bsAb binds to the T cell with one binding arm (e.g. CD3 or CD2)
and activates it at the same time, co-stimulatory signals from the Fc receptor-positive
cell bound to the Fc portion of the bsAb can be transferred to the T cell. That means,
only the combination of T cell activation via one binding arm of the bsAb and simultaneous
mediation of co-stimulatory signals from the Fc receptor-positive cell to the T cell
leads to efficient T cell activation.
[0031] Also tumor-specific T cells which have been insufficiently activated at the tumor
cell site and, therefore, remain in an anergic state may be reactivated.
[0032] A physiological activation of tumor-retargeted T cells is achieved by 1) binding
of the bsAb to the T cell, for example via the T cell receptor complex, and 2) simultaneous
transfer of costimulatory signals by FcR+ cells binding to the Fc portion of the bsAb.
[0033] Thus, an important prerequisite for an efficient reduction of GVHD is the use of
trAbs having a Fc portion able to bind to FcγR+ cells which are activated themselves
by this event and, thereby, able to mediate co-stimulatory signals to the T cell in
combination with the redirection of effector cells away from healthy host tissue towards
tumor cells.
[0034] Due to this mechanism, tumor-specific anergic T cells (having a T cell receptor [TCR]
which recognize tumor-specific peptides in association with MHC molecules on the tumor
cell) can be reactivated at the tumor cell site, and thereby tumor tolerance can be
reversed.
[0035] Since the tumor-specific T cells via their TCR are able to recognize completely different
peptides/proteins than the trAb via its anti-tumor binding arm, additional tumor cells
may be recognized and killed after activation of such T cells by the trAb. That means,
it is not neccessary for the trAb to recognize all of the tumor cells which in a second
step subsequently can be eliminated by activated T cells with an adequate T cell receptor.
[0036] Preferably, the antibodies according to the invention are monoclonal, chimeric, recombinant,
synthetic, semi-synthetic, or chemically modified intact antibodies having for example
Fv, Fab, scFv, or F (ab)2 fragments.
[0037] In the method of the present invention also antibodies or derivatives or fragments
of human origin can be used, or antibodies modified to be suitable for the use in
humans (so-called "humanized antibodies") (see for example Shalaby et al., J. Exp.
Med. 175 (1992), 217; Mocikat et al., Transplantation 57 (1994), 405).
[0038] The preparation of the different types of antibodies and antibody fragments mentioned
above is obvious to the skilled artisan. The preparation of monoclonal antibodies
preferably of mammalian origin, e.g. of human, rat, mouse, rabbit, or goat, can be
performed using conventional methods for example as those described in Köhler and
Milstein (Nature 256 (1975), 495), in Harlow and Lane (Antibodies, A Laboratory Manual
(1988), Cold Spring Harbor) or in Galfré (Meth. Enzymol. 73 (1981), 3).
[0039] It is further possible to prepare the antibodies described by means of recombinant
DNA technology according to techniques obvious to the skilled artisan (see Kurucz
et al., J. Immunol. 154 (1995), 4576; Hollinger et al., Proc. Natl. Acad. Sci. USA
90 (1993), 6444).
[0040] The preparation of antibodies having two different specificities, the so-called bispecific
antibodies, can be performed for example using recombinant DNA technology but also
by the so-called hybrid hybridoma fusion technique (see for example Milstein et al.,
Nature 305 (1983), 537). This technique consists of fusing hybridoma cell lines each
producing antibodies having one of the desired specificities and identifying and isolating
recombinant cell lines producing antibodies having both specificities.
[0041] The problem forming the basis of the invention can be overcome using either trifunctional
bispecific or trispecific trifunctional antibodies if they exhibit the properties
and effects as described herein. The invention is particularly described by the way
of trifunctional bispecific antibodies. However, it is understood that it also covers
the following trispecific antibodies exhibiting similar effects.
[0042] The preparation of antibodies exhibiting three specificities, so-called trispecific
antibodies, also suitable to solve the basic problem of the invention may for example
be carried out by coupling a third antigen binding site having an additional specificity,
e.g. in the form of "single chain variable fragments" (scFv) to one of the IgG heavy
chains of a bispecific antibody. The scFv may be coupled for example using a-S-S(G4S)nD-I(SEQ
ID NO:1)linker to one of the heavy chains (S=serine, G=glycine, D=aspartate, I=isoleucine).
[0043] Analogously, trispecific F(ab)2 constructs may be prepared by replacing the CH2-CH3
regions of the heavy chain of one specificity of a bispecific antibody by an scFv
having a third specificity, while the CH2-CH3 regions of the heavy chain having the
other specificity can be removed for example by insertion of a stop codon (at the
end of the "hinge" 5 region) into the coding gene, e.g by homologous recombination.
[0044] It is also possible to prepare trispecific scFv constructs wherein three VH-VL regions
representing three different specificities are arranged in series.
[0045] According to the invention there are intact bispecific antibodies used. Intact bispecific
antibodies are composed of two antibody semi-molecules (each having a H and a L immunoglobulin
chain) each representing a specificity, and additionally like normal antibodies having
a Fc portion performing the well-known effector functions. They are preferably prepared
using the quadroma technology. This method of preparation is exemplified in DE-A-44
19 399. For complete disclosure this document is incorporated in its entirety by reference
also with respect to a definition of bispecific antibodies. It should be understood
that other methods of preparation are also useful if they lead to the intact bispecific
antibodies according to the above definition required according to the invention.
[0046] For example, intact bispecific antibodies may be produced in sufficient amounts using
a newly developed method of preparation (Lindhofer et al., J. Immunology, 155:219
(1995)). The combination of two bispecific antibodies directed against two different
tumor-associated antigens (e.g. c-erb-B2, EpCAM, such as GA-733-2=C215) on the mammary
carcinoma cells minimizes the risk that tumor cells expressing only one of the antigens
remain unidentified.
[0047] Bispecific antibodies are able to bind to the T cell receptor complex on the T cell
with one binding arm and to tumor-associated antigens on the tumor cell with the second
binding arm. Thereby, T cells are activated which kill the tumor cells by releasing
cytokines or by apoptosis-mediating mechanisms. In addition, there is the possibility
that T cells recognize tumor-specific antigens by their receptor during activation
by the bispecific antibodies and, thereby, a long-lasting immunization is initiated.
Of particular importance in this respect is the intact Fc portion of the bispecific
antibody which mediates the binding to accessory cells such as monocytes/macrophages/dendritic
cells and causes these cells to develop cytotoxicity, and/or concomitantly transfer
important co-stimulatory signals to the T cell.
[0048] The antibodies according to the invention uses preferably intact bsAb able to bind
to Fc[gamma]RI+ cells via their Fc portion. In addition, the bsAb used according to
the invention have as a second specificity besides the tumor cell-binding specificity
for example anti-CD3, i.e. they are able to bind to T cells via the second binding
arm. Thus, by the bsAb used according to the invention T cells are activated and redirected
against the tumor cells.
[0049] The present invention comprises two main embodiments:
- 1. In the first embodiment, the patient to be treated has undergone an allogeneic
bone marrow transplantation or an allogeneic stem cell transplantation. After having
been transplanted, the patient will be administered with the allogeneic effector cells
together with the trifunctional bispecific and/or trispecific antibodies as indicated
herein. Administration of the allogeneic effector cells is also referred to as donor
lymphocyte infusion (DLI). DLI refers to a method of treating cancer in which effector
cells of lymphoid and/or myeloid origin are transferred, particularly by infusion,
into the person who received the original bone marrow or stem cell transplant.
- 2. In a second embodiment of the invention, allogeneic effector cells in combination
with said trifunctional bispecific and/or trispecific antibodies can be administered
in a pharmaceutical preparation by the way of allogeneic donor lymphocyte infusion
into patients which received a mild immunosuppression or conditioning (by an e.g.
sublethal radiation dose) without having received an allogeneic bone marrow or stem
cell transplantation before. In this embodiment, the conditioning allows the infused
allogeneic effector cells to survive for a time period which is necessary to accomplish
tumor cell killing before rejection. That means that a host versus graft reaction
will not immediately attack and destroy the effector cells of the DLI. Moreover, such
a variation would further minimize the risks of an allogeneic bone marrow or stem
cell transplantation which currently are limiting a broader application of this therapy.
[0050] DLI comprises the infusion of lymphocytes from a bone marrow donor into a person
who received the original transplant. Donor lymphocyte infusion has mainly been used
to treat relapsed chronic myelogenous leukemia (CML). Patients with relapsed acute
leukemia, chronic lymphocytic leukemia (CLL), myelodysplasia (MDS), Hodgkin disease,
non-Hodgkin lymphoma (NHL), and multiple myeloma have also been treated by DLI.
[0051] The allogeneic effector cells and the trifunctional bispecific antibodies and/or
trispecific antibodies can be either administered to the patient in need thereof separately
or preferably in admixture. In a preferred method of the invention, the effector cells
and the antibodies are pre-incubated for a time period of up to two hours, preferably
for a time between 5 minutes and 30 minutes, most preferably about 2 hours. Further
preferred is a time limit of about 1 minute to 10 minutes.
[0052] A person skilled in the art, particularly a physician working in the field of immunology,
will know about the conditions which are more preferably to be used and how to adapt
them to a particular patient and disease. It is state of the art to make appropriate
variations and adaptions to the present method in order to provide the best benefit
for the patient to be treated.
[0053] The most characteristic feature of the malignant tumor treatment method and composition
according to the invention is that a malignant tumor can be treated while preventing
or avoiding or at least sufficiently reducing the onset of GVHD as an adverse effect,
which is one of the most important drawbacks of the technique of DLI.
[0054] The malignant tumor which can be treated by the method of this invention includes
malignant tumors of hematopoietic cells, such as leukemia, malignant lymphoma, and
multiple myeloma; malignant tumors other than those of the hematopoietic cells, for
example melanoma, sarcoma, and brain tumor; and all organ cancers (solid cancers)
such as stomach cancer, tongue cancer, esophageal carcinoma, colorectal cancer, liver
cancer, gallbladder carcinoma, pancreatic carcinoma, renal carcinoma, bladder cancer,
nasopharyngeal cancer, laryngeal cancer, skin cancer, mammary cancer, testicular cancer,
ovarian cancer, uterus carcinoma, and lung cancer.
[0055] In the second method of the invention, DLI is first performed in a patient with malignant
tumor to replace transiently the hematopoietic system of the patient with that of
a donor. The thus-constructed donor-derived hematopoietic system (mainly T lymphocytes)
attacks the tumor cells of the patient in the manner of GVT, damaging and killing
them and producing a curative effect on the tumor. On the other hand, the donor-derived
hematopoietic system regards normal tissues of the patient as a foreign matter and
causes GVHD to eliminate the same. In accordance with the invention, however, together
with allogeneic effector cells contained in DLI the trifunctional antibodies of the
present invention as defined above are infused in a patient in need thereof whereby
the onset of GVHD can be prevented or at least substantially reduced.
[0056] The use and composition of the invention make it possible to inhibit or avoid or
effectively lower GVHD as an adverse effect possibly induced by DLI and cure various
sorts of malignant tumor by preferably repeating DLI several times in combination
with trifunctional antibodies by which in order to erase malignant tumor growth and
reoccurrence.
[0057] The DLI employed in accordance with the invention can be performed basically in the
same manner as the conventional DLI in recurrence therapy for leukemic cells (cf.
Shintaro Shiobara, Hematology & Oncology, 42(2):151-157, 2001).
[0058] The donor is preferably a normal or related subject. Further, the donor is allogeneic
to the patient (host), to whom the method of the invention is to be applied. The histocompatibility
antigens (HLA) of the donor can be matched or mismatched in several loci compared
to the recipient.
[0059] The donor lymphocytes to be used in DLI are included in cell collections (preparations)
selected from peripheral blood mononuclear cells (PBMCs), whole bone marrow cells,
splenic cells or other sources as e.g. cord blood derived from the allogeneic donors
mentioned above. The collection and preparation of PBMCs can be performed e.g. by
apheresis or bone marrow puncture.
[0060] The allogeneic effector cells used in the DLI and contained in the apheresate are
mainly of lymphoid and myeloid origin comprising accessory cells as e.g. monocytes,
macrophages, dendritic cells and natural killer cells. These effector cells express
Fcγ receptors of type I, II and III. Moreover, all types of T cells can be present.
[0061] In the practice of the invention, DLI is carried out preferably in the manner of
intravenous infusion of e.g. PBMCs. In particular, those PBMC preparations obtained
by the ordinary method contain T cells at a level of about 20% or higher.
[0062] The amount of e.g. PBMCs to be transfused and the frequency of transfusions can be
appropriately determined depending on the condition (age, sex, body weight) of the
patient (host) and the severity of disease, among others, without any particular restriction.
Generally, the amount is usually selected within the range of about 5x10e6 to 1x10e8
cells/kg per shot. The frequency of transfusions and the interval thereof may be such
that the desired GVT-based curative effect on malignant tumor can be produced. Generally,
at least two, usually about 3-5, transfusions are performed usually at intervals of
4-60 days.
[0063] For increasing the treatment efficiency of this DLI on malignant tumor, the method
of the invention may further comprise subjecting the host to irradiation treatment
as a pretreatment prior to DLI and antibody application. This irradiation treatment
should destroy as much lymphatic system and hematopoietic cells of the host as necessary
to mitigate or avoid host versus graft reaction. Generally, total body irradiation
at a dose of at most about 4-5 Gy is sufficient. This total body irradiation is preferably
performed on the same day (within 24 hours) as the day of DLI (first time).
[0064] The present invention also provides a pharmaceutical composition for performing the
above-described method for the treatment of malignant tumor growth and metastasis.
The composition comprises a composition containing donor-derived allogeneic effector
cells and trifunctional antibodies as defined herein in a therapeutically effective
amount, preferably in combination with pharmaceutically acceptable carries, diluents,
auxiliary substances etc.
[0065] Generally, those compositions are prepared preferably in the form suited for the
route of administration thereof, for example in the form of injections, transfusions
or like liquids or solutions. The liquid or solution forms, inclusive of injections,
can be prepared in the same manner as in preparing various conventional pharmaceutical
preparations containing cell components of this kind. The carrier to be used on that
occasion may be any of various pharmaceutically acceptable carriers (diluents) so
far well known in this field of art. Specific examples thereof are physiological sodium
chloride solution, PBS and RPMI 1640. In preparing the above-mentioned liquid or solution
forms, various technologies currently in general use in preparing various transfusions
can be used. The respective compositions may be prepared just prior to use. The respective
compositions are administered at respective predetermined doses via a predetermined
route(s) of administration according to the method of the invention.
[0066] The DLI employed in accordance with the invention can be performed basically in the
same manner as the conventional DLI in recurrence therapy for leukemic cells (cf.
Shintaro Shiobara, Hematology & Oncology, 42(2):151-157, 2001).
[0067] The problem of the present invention has been solved as demonstrated in the following
experiments. The example is to be understood to exemplify the invention; the invention
is however not restricted to this particular embodiment. A person skilled in the art
will be able to make the necessary modifications and adaptations within the spirit
of this invention.
Example 1
Materials and methods
Mice
[0068] Female BALB/c H-2
d (BALB), C57BL/6 H-2
b (C57) and (BALB x C57BL/6)F
1 H-2
d/b (F
1) mice aged 10-12 weeks and weighing 22-24 grams, were used in this study. All mice
were purchased from Harlan, Israel, and maintained in the animal facility of the Hadassah
University Hospital with sterilized food and water ad libitum, in full compliance
with the regulations for the protection of animal rights.
Tumor cells
[0069] A murine model of melanoma cell line (B-16) transfected with human EpCAM was used
in the experiments (7). The tumor cells were maintained in RPMI 1640 medium supplemented
with 5% fetal bovine serum (FBS) (GIBCO, N.Y., USA), 2mM L-glutamine, 1% non-essential
amino acids, 1mM sodium pyruvate, 0.5mg/ml geneticin (GIBCO, N.Y., USA) 100U/ml penicillin,
and 100µg/ml streptomycin. All culture supplements (except FBS and geneticin) were
purchased from Biological Industries, Beit HaEmek, Israel. Cells were kept at 37°
C in a humidified 5% CO
2 air incubator. B-16-EpCAM cells were harvested by 0.25% trypsin in 0.05% EDTA, washed
with RPMI 1640 and resuspended for intraperitoneal (IP) inoculation (0,25 ml/mouse).
Spleen cells
[0070] Splenocytes were prepared by teasing spleen cells over a metal mesh, then filtering
them through nylon mesh and suspending them in phosphate buffered saline (PBS) for
IP injection (30X10
6/mouse).
Radiation Therapy
[0071] Sublethal total body irradiated (TBI) was administered using a 6MEV linear accelerator
at a dose rate of 1.9Gy/min (400cGy) in order to prevent rejection of donor spleen
cells used for induction of GVHD and graft-versus-tumor (GVT) effects.
Antibody treatment
[0072] The trifunctional bispecific antibody (BiLu) consisting of specific antigen binding
regions directed against murine CD3 and human EpCAM (20) was used in the experiments.
BiLu was given intraperitoneal (IP) either alone or in conjunction with cell therapy
(10µg/mouse).
Experimental design
[0073] Recipient F
1 mice were conditioned with non-lethal TBI of 4 Gy. Twenty-four hours later, 5X10
3 or 5X10
4 B-16-EpCAM tumor cells were inoculated IP. On the following day, 30X10
6 naive spleen cells derived from C57 or BALB donors were incubated with BiLu antibodies
for 10 min and then the mixture was inoculated IP.
Follow-up
[0074] In all experiments, mice were checked daily for the appearance of signs and symptoms
of GVHD such as hunched posture, ruffled fur, diarrhea and cachexia as assessed by
weight. Body weight was measured on a weekly basis. Survival was monitored and GVHD-related
death was determined if mice were showing GVHD symptoms as described above. In addition,
mice were investigated post mortem for detection of the presence of tumor metastases
in the peritoneum, in order to determine tumor- related death.
Flow cytometry analysis
[0075] B16-EpCAM tumor cells (4X10
5) were incubated with 0.5µg BiLu antibody in staining buffer (PBS containing 1% bovine
serum albumin and 0.03% sodium azide) for 30min. on ice, then washed and incubated
with FITC F(ab')2 fragment mouse anti-rat IgG (H+L) (Jackson ImmunoResearch Laboratories,
INC, Pa. USA) for 30min on ice. Tumor cells were analyzed by FACS (FACStar plus; Becton-Dickinson,
Ca, USA) and the percentage of tumor cells expressing EpCAM determinant was measured.
In all tests > 88% B16-EpCAM cells stained positive (data not shown).
Statistical analysis
[0076] Body weights were presented as mean ± SE (standard error). The Kaplan-Meier method
was used to calculate the probability of survival as a function of time after tumor
inoculation. The statistical significance of survival between pairs of Kaplan-Meier
curves was evaluated by the log rank test. Statistical significance of differences
in body weights of control versus BiLu treated mice was evaluated by standard two-tailed,
unpaired student t-test. A value of p<0.05 was considered statistically significant.
Results
The effect of BiLu treatment on GVHD symptoms
[0077] GVHD was induced by intravenous inoculation of naive splenocytes derived from C57
donors in sublethally irradiated F
1 mice that had been inoculated with 5X10
3 B16-EpCAM tumor cells. A high percentage of mice inoculated with naive C57 splenocytes
(23/27) displayed GVHD symptoms such as hunched posture, ruffled fur, diarrhea and
loss of 2 grams of body weight over 18 days (Figure 1). On the other hand, only 5/25
mice inoculated with BiLu pretreated C57 splenocytes displayed GVHD symptoms. Most
of the mice inoculated with C57 cells pretreated with BiLu, gained 2-3 grams in weight
over 18 days (Figure 1) and maintained a normal healthy appearance for >170 days.
Differences in body weight of untreated as compared with BiLu pretreated naive splenocytes
obtained from C57 mice on days 10 and 18 was statistically significant (p<0.05).
[0078] One injection of naïve BALB splenocytes into F
1 mice inoculated with B16-EpCAM tumor cells, caused mild GVHD symptoms, such as hunched
posture and ruffled fur; After a second administration of BALB splenocytes these symptoms
were more prominent although there was no significant loss of body weight (data not
shown) and most of the mice finally recovered. In contrast, mice inoculated with BALB
splenocytes pretreated with BiLu antibodies, did not display any signs or symptoms
of GVHD during a follow-up period of >250 days and also showed no signs of tumor growth.
[0079] The GVT effect of host allogeneic lymphocytes and trifunctional bispecific antibody
C57 splenocytes, which are syngeneic to the B16-EpCAM tumor cells but haploidentically
mismatched to the host cells, were inoculated into sub-lethally irradiated F
1 mice with or without BiLu pretreatment. Control F
1 mice injected with 5X10
3 cells tumor cells and inoculated with C57-derived naïve splenocytes, died of GVHD
(23/27) after a median of 20 days, while GVHD- related death in F
1 mice treated with BiLu pretreated naïve C57 splenocytes was observed only in 5/25
(p=0.000). Sixteen out of 25 mice treated with BiLu pretreated naïve cells, remained
tumor-free with no signs of GVHD for a median of >212 days (Table 1).
Table 1. Effect of trifunctional bispecific antibody (BiLu) on GVHD and GVT induction
by C57BL/6 spleen cells in mice inoculated with 5 x103 B16 EpCAM melanoma cells
Effector cells (C57BL/6) |
BiLU pretreatment (10µg/ml) |
Survival (days) Median (range) |
Cause of Death |
Disease free survivors |
Tumor |
GVHD |
- |
- |
33 (24->212) |
60 |
- |
7 |
|
|
|
} p=0.000 |
|
} p=0.000 |
|
+ |
>212 (24->212) |
3 |
- |
7 |
Splenocytes |
- |
20 (14->212) |
3 |
23 |
1 |
|
|
|
}p=0.005 |
}p=0.000 |
}p=0.000 |
Splenocytes |
+ |
>212 (25->212) |
4 |
5 |
16 |
[0080] (BALB/c/cXC57BL/6)F
1 mice were inoculated with 5 x 10
3 B16-EpCAM melanoma cell 24h after conditioning with sublethal TBI (4Gy). One day
later, 30 x 10
6 naive C57BL/6 splenocytes were given intraperitoneally with or without pretreatment
with BiLu (10µg/mouse). p values are shown for the comparison of GVT and GVHD with
or without BiLu pretreatment of C57BL/6 naïve cells.
[0081] BiLu treatment without cell therapy had an anti-tumor effect on mice inoculated with
a low tumor cell dose of 5X10
3. Its efficacy in preventing GVHD, in tumor-bearing mice supposedly by targeting the
donor T cells to the tumor, encouraged us to test its effect in mice inoculated with
a higher tumor cell dose (5X10
4), proved lethal in 100% of untreated mice (68/68) with a median survival of 21 days.
Although BiLu treatment without cell therapy had also a substantial anti-tumor effect
on the high tumor cell dose (10/17 mice remained tumor-free), BiLu treatment given
concomitantly with allogeneic cell therapy using C57 naive splenocytes, resulted in
10/20 healthy appearing mice with no GVHD and no evidence of tumor for >250 days.
Treatment with BiLu, protected recipients of alloreactive C57 spleen cells as GVHD-related
death was observed in only 4/20 mice inoculated with naive C57 cells, whereas 31/32
untreated control mice inoculated with untreated naive cells died of GVHD after a
median of 19 days (Table 2).
Table 2. Effect of trifunctional bispecific antibody (BiLu) on GVHD and GVT induction
by C57BL/6 spleen cells in mice inoculated with 5 x 104 B16 EpCAM melanoma cells
Effector cells (C57BL/6) |
BiLU pretreatment (10µg/ml) |
Survival (days) Median (range) |
Cause of Death |
Disease free survivors |
Tumor |
GVHD |
- |
- |
21 (17-34) |
68 |
- |
0 |
|
|
|
} p=0.000 |
|
} p=0.000 |
- |
+ |
>220 (30->220) |
7 |
- |
10 |
Splenocytes |
- |
19(14-39) |
1 |
31 |
0 |
|
|
|
} p=0.006 |
}p=0.000 |
}p=0.000 |
Splenocytes |
+ |
>290 (26->290) |
6 |
4 |
10 |
[0082] (BALB/c/cXC57BL/6)F
1 mice were inoculated with 5 x 10
4 B16-EpCAM melanoma cell 24h after conditioning with sublethal TBI (4Gy). One day
later, 30 x 10
6 naïve C57BL/6 splenocytes were given intraperitoneally with or without pretreatment
with BiLu (10µg/mouse). p values are shown for the comparison of GVT and GVHD with
or without BiLu pretreatment of C57BL/6 naive cells.
The effect of BiLu treatment on allogeneic cell therapy
[0083] BALB splenocytes which are fully mismatched to B16-EpCAM tumor cells but haploidentically
mismatched to host alloantigens, were inoculated into sub-lethally irradiated F
1 mice with or without prior pretreatment with BiLu. One dose of BALB splenocytes in
mice inoculated with 5X10
3 tumor cells neither produced a graft versus tumor (GVT) effect (18/32 mice died of
tumor) nor caused lethal GVHD (only 1/32 mice died of GVHD) (Table 3). Two doses of
BALB splenocytes, however, induced GVT effect accompanied with severe GVHD, resulting
in 6/27 tumor-related deaths, 7/27 GVHD related death and 14/27 disease free survivors.
Mice inoculated with 5X10
3 tumor cells and treated with BiLu and one or two doses of BALB splenocytes, led to
19/20 and 10/10 tumor- and GVHD-free mice respectively, after a follow-up period of
>212 days (Table 3).
Table 3. Effect of trifunctional bispecific antibody (BiLu) on GVHD and GVT induction
by BALB/c spleen cells in mice inoculated with 5 x 103 B16 EpCAM melanoma cells
Effector cells (BALB/c) |
BiLu pretreatment (10µg/ml) |
Survival (days) Median (range) |
Cause of Death |
Disease free survivors |
Tumor |
GVHD |
- |
- |
33 (24-212) |
60 |
- |
7 |
|
|
|
} p=0.000 |
|
} p=0.000 |
- |
+ |
>212 (24->212) |
3 |
- |
7 |
1Splenocytes |
- |
66 (26->212) |
18 |
1 |
13 |
|
|
|
} p=0.000 |
|
} p=0.000 |
|
|
|
|
}p=0.0244 |
|
Splenocytes |
+ |
>212 (43->212) |
1 |
0 |
19 |
2Splenocytes |
- |
>196 (170->212) |
6 |
7 |
14 |
|
|
|
}p=0.109 |
}p=0.031 |
} p=0.007 |
Splenocytes |
+ |
>212 |
0 |
0 |
10 |
[0084] (BALB/c x C57BL/6)F
1 mice were inoculated with 5 x 10
3 B16-EpCAM melanoma cell 24h after conditioning with sublethal TBI (4Gy). Naive 1
(1) or 2
(2) doses BALB splenocytes (30 x 10
6) were administered intraperitoneally with or without pretreatment with BiLu (10µg/mouse)
one day later. Cell therapy with 2 doses versus 1 dose of BALB splenocytes, both without
BiLu, significantly (p=0.006) reduced number of tumor related death, significantly
(p=0.039) increased number of GVHD related death, but was not statistically different
for the disease free survivors (p=0.186).
Table 4. Effect of trifunctional bispecific antibody (BiLu) on GVT and GVHD in mice
inoculated with 5 x 104 B16 EpCAM melanoma cells
Effector cells BALB/c |
BiLU pretreatment (10µg/ml) |
Survival (days) Median (range) |
Cause of Death |
Disease free survivors |
Tumor |
GVHD |
- |
- |
21 (17-34) |
68 |
- |
0 |
|
|
|
} p=0.000 |
|
} p=0.000 |
|
+ |
>220 (30->220) |
7 |
- |
10 |
Splenocytes |
- |
52 (17->245) |
26 |
0 |
6 |
|
|
|
} p=0.002 |
} p=0.541 |
p=0.000 |
Splenocytes |
+ |
>286 (22->286) |
8 |
1 |
13 |
[0085] (BALB/c x C57BL/6)F
1 mice were inoculated with 5 x 10
4 B16-EpCAM melanoma cell 24h after conditioning with sublethal TBI (4Gy). One day
or/and 5 days following tumor inoculation, 30 x 10
6 BALB splenocytes were given intraperitoneally with or without pretreatment with BiLu
(10µg/mouse).
[0086] The effect of BiLu treatment on GVHD induction in tumor-free in comparison with mice
inoculated with B16-EpCAM tumor cells.
[0087] Since BiLu treatment was very efficient in preventing GVHD in tumor- bearing mice,
it was interesting to test its effect on GVHD induction in tumor-free mice. Results
presented in Figure 2 show that injection of haploidentically mismatched C57 splenocytes
into F
1 mice that had not been inoculated with tumor cells induced lethal GVHD in 18/18 animals.
Pretreatment of naïve C57 cells with BiLu did not prevent GVHD-related death in 5/5
mice. The median survival of these mice was 19 and 24 days respectively, in comparison
with median survival of 76 and 30 days for 5/25 mice and for 4/20 mice inoculated
with 5X10
3 or 5X10
4 tumor cells and treated with BiLu, respectively. Statistical analysis revealed that
pretreatment of the GVHD inoculum with BiLu in mice inoculated with 5X10
3 or 5X10
4 tumor cells produced a significantly better anti-GVHD effect in comparison with similarly
treated tumor-free recipients inoculated with BiLu pretreated C57 cells (p=0.000).
[0088] It could be shown by the experiments described above that trifunctional bispecific
antibodies, BiLu, e.g. directed against human tumor antigen (EpCAM) and murine CD3,
given with lymphocytes fully alloreactive against the host, successfully prevented
lethal GVHD while exerting an efficient anti-tumor effect, leading to disease-free
survival of >250 days in mice inoculated with EpCAM transduced B16 melanoma. The anti-GVHD
effect of BiLu was especially evident in sublethally irradiated F
1 mice inoculated with parental C57 splenocytes. Since the intensity of GVHD induced
by a similar dose of parental naive BALB splenocytes was far less dramatic, as has
already been discussed elsewhere, the anti-GVHD effect of BiLu in this setting could
not be evaluated. However, when two doses of BALB derived splenocytes were given,
and more severe GVHD developed, the anti-GVHD effect of BiLu was clearly evident.
[0089] Our main goal was to determine the possible role of tumor existence on targeting
the alloreactive lymphocytes by BiLu to the tumor in order to achieve the most efficient
GVT effect while controlling or minimizing GVHD development.
[0090] Remarkably, the anti-GVHD effect of BiLu was clearly depended on the presence of
tumor cells bearing the EpCAM antigen in the recipient (figure 2). While even a low
dose of tumor cells was sufficient to confer significant protection from lethal GVHD
in 80% of the mice, BiLu did not prevent GVHD induction in naïve non-tumor bearing
F
1 mice inoculated with C57 splenocytes (figure 2). It is reasonable to assume that
in the absence of tumor, BiLu was ineffective in preventing the donor cells from targeting
GVHD-sensitive organs in the host. In view of another report showing that in the absence
of tumor target, BsAb treatment by itself does not activate systemic stimulation of
T cells, one may assume that the GVHD that occurred in non-tumor bearing F
1 hosts was mediated primarily by the inoculated spleen cells themselves, without the
risk of engagement of BiLu activated host cells.
[0091] As shown here and as reported previously (20), BiLu treatment alone given after inoculation
of a low dose of tumor cells can provide an efficient anti-tumor effect.
[0092] The anti-tumor effect mediated by C57 splenocytes alone could not be evaluated due
to the incidence of severe and lethal GVHD. In contrast, some beneficial GVT effect
(13 of 32 mice survived, table 3) could be observed after treatment with naïve BALB
splenocytes without additional BiLu, since GVHD was mild and non-lethal. This anti-tumor
effect was further amplified (14 of 27 mice survived, table 3) by inoculation of 2
consecutive doses of BALB splenocytes (p ≤0.006) for comparison with one dose of BALB
or, as a control, of tumor alone. The use of alloCT by itself has previously been
shown to be an effective immunotherapeutic strategy for achieving GVL/GVT effects
in experimental animal models of leukemia and mammary carcinoma, as well as in patients
with hematological malignancies and metastatic solid tumors (26). However, GVT effects
against tumor cells were dramatically improved by the addition of BiLU treatment (10
of 10 mice survived, table 3) which enhanced the targeting of alloreactive effector
cells, while diverting such cells away from host tissues susceptible to GVHD. Remarkably,
no mouse was lost due to GvHD in the BiLu group whereas 7 out of 27 mice died of GvHD
and 6 of 27 due to tumor growth, after 2 splenocyte infusions in the non-BiLu group.
[0093] Due to the trifunctional properties of the BiLu (CD3xEpCAM) used in our study, it
was feasible to target either naive splenocytes containing CD3
+ T cells or FcγR
+ NK cells, as well as monocytes and antigen-presenting dendritic cells, all of which
may contribute to the achievement of a most effective anti-tumor response. Since in
many primary tumor cells, and more frequently in metastatic cells, the expression
of class I MHC antigens is either lost or suppressed, the combination of T cell dependent
and the non-MHC- restricted killing activity exerted by accessory cells, may circumvent
aberrant down-regulated MHC expression by tumor cells.
[0094] The BiLu antibodies directed to CD3 and EpCAM antigens serve as a relevant model
for a variety of e.g. epithelial tumors over-expressing EpCAM or Her2/neu or other
malignancies expressing further tumor-associated antigens as target antigens on their
surface (13,15,17).
[0095] Although in principle, bispecific antibodies can be used alone, through activation
of the patients own cells, the combination of such constructs with alloCT would make
it possible to benefit from different anti-cancer effector mechanisms in order to
achieve maximal targeting of anti-cancer effector cells to the tumor site, while in
parallel, avoiding or minimizing the risk of uncontrolled GVHD.
[0096] Summarizing, bispecific monoclonal antibodies (BiLu) directed against murine CD3
and a tumor-associated antigen of human epithelial cell adhesion molecule (EpCAM)
were tested for their ability to induce cell-mediated adoptive immunotherapy in a
murine melanoma (B-16) model of C57BL/6 (C57) mice transfected with EpCAM. Naive or
rIL-2 activated splenocytes (LAK) induced lethal graft versus host disease (GVHD)
in 64-97% of sublethally irradiated (BALB/cXC57BL/6)F
1 (F
1) hosts inoculated with a sub-lethal (5X10
3) or lethal (5X10
4) dose of tumor cells. BiLu antibodies given concomitantly with alloreactive C57 cells,
effectively prevented GVHD-related-and tumor-related death in 16/25 and 8/12 mice
inoculated with 5X10
3 tumor cells and treated with naive or LAK C57 cells, respectively, as well as in
10/20 and 22/27 mice inoculated with 5X10
4 tumor cells treated with naive or LAK C57 cells, respectively, over a follow-up period
of >250 days. Naïve or LAK cells derived from BALB/c (BALB) mice caused mild GVHD
which was lethal in only 0-27% of the F
1 mice, while they exerted an efficient graft versus tumor effect which was further
improved by BiLu treatment given concomitantly with naive but not with LAK BALB splenocytes,
probably due to excessive activation of BALB against the fully MHC mismatched tumor
cells. Bispecific antibodies capable of cross-linking T lymphocytes, natural killer,
and other FcγR
+ effector cells to the tumor cells, may be applied together with adoptive allogeneic
cell therapy to maximize anti-tumor responses while acting on GVHD in patients with
minimal residual disease.
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1. Use of allogeneic effector cells together with trifunctional bispecific or trispecific
antibodies having the following properties:
a. binding to a T cell
b. binding to at least one antigen on a tumor cell
c. binding via their Fc portion in the case of trifunctional bispecific antibodies
or via a third specificity in the case of trispecific antibodies to Fc receptor positive
cells
said antibodies redirecting the allogeneic cells away from host tissues in order to
substantially reduce or avoid a graft versus host disease for the manufacture of a
medicament for treating tumor growth and metastasis in a mammal.
2. Use according to claim 1, wherein said allogeneic effector cells are lymphoid or myeloid
cells or a combination thereof.
3. Use according to claim 1 or 2, wherein said allogeneic effector cells are lymphoid
cells selected from the group consisting of T cells, NK cells and B cells or a combination
thereof.
4. Use according to one or more of the preceding claims, wherein said allogeneic effector
cells are myeloid cells selected from the group consisting of monocytes, macrophages,
and dendritic cells or a combination thereof.
5. Use according to one or more of the preceding claims, wherein said allogeneic effector
cells are lymphoid or myeloid cells or a combination thereof used in the form of peripheral
blood mononuclear cells, whole bone marrow cells, cord blood and splenic cells.
6. Use according to one or more of the preceding claims, wherein said allogeneic effector
cells are derived from a donor, matched or mismatched in HLA type to the host.
7. Use according to one or more of the preceding claims, wherein before administering
said allogeneic effector cells together with said antibodies said mammal is treated
in order to reduce its tumor load.
8. Use according to one or more of the preceding claims, wherein said mammal had not
received a hematopoietic stem cell or allogeneic bone marrow transplantation and wherein
before administering said allogeneic effector cells together with said antibodies
said mammal is treated in order to induce a mild immunosuppression or conditioning
to allow said administered allogeneic effector cells to survive for a time period
which is necessary to accomplish tumor cell killing by said antibodies before a rejection
reaction against said effector cells is initiated.
9. Use according to one or more of the preceding claims, wherein said treatment to induce
a mild immunosuppression or conditioning is performed by irradiation.
10. Use according to one or more of the preceding claims, wherein before administering
said allogeneic effector cells together with said antibodies said mammal had undergone
hematopoietic stem cell transplantation or bone marrow transplantation.
11. Use according to one or more of the preceding claims, wherein before administering
said allogeneic effector cells together with said antibodies said mammal is treated
in order to reduce its tumor load by irradiation, chemotherapy, immune therapy, surgery
or a combination thereof.
12. Use according to one or more of the preceding claims, wherein said trifunctional antibodies
are bi-specific antibodies selected from the group consisting of anti-CD3 X anti-tumor-associated
antigen antibody, anti-CD2 X anti-tumor-associated antigen antibody, anti-CD5 X anti-tumor-associated
antigen antibody, anti-CD28 X antitumor-associated antigen antibody, and anti-CD44
X anti-tumor-associated antigen antibody.
13. Use according to one or more of the preceding claims, wherein said trifunctional antibodies
are heterologous rat/mouse bi-specific antibodies.
14. Use according to one or more of the preceding claims, wherein said trifunctional antibodies
are mouse IgG2a/rat IgG2b heterologous bi-specific antibodies.
15. Use according to one or more of the preceding claims, wherein said at least one antigen
on a tumor cell recognized by said trifunctional antibodies is one of EpCAM, Her2/neu,
EGF-receptor, GD2, GD3, G250, CEA, CD20, CD22, CD30, CD33, CD38 or CD138.
16. Use according to one or more of the preceding claims, wherein said tumor is a malignant
tumor selected from leukemia, malignant lymphoma chronic myelogenous leukemia (CML),
acute leukemia, chronic lymphocytic leukemia (CLL), myelodysplasia (MDS), Hodgkin
disease, non-Hodgkin lymphoma (NHL), multiple myeloma, sarcoma, melanoma, brain tumor,
stomach cancer, tongue cancer, esophageal carcinoma, colorectal cancer, liver cancer,
gallbladder carcinoma, pancreatic carcinoma, renal carcinoma, bladder cancer, nasopharyngeal
cancer, laryngeal cancer, skin cancer, mammary cancer, testicular cancer, ovarian
cancer, uterus carcinoma, and lung cancer.
17. Use according to one or more of the preceding claims, wherein said antibodies are
used in an amount of 10-100µg/patient and treatment.
18. Use according to one or more of the preceding claims, wherein the mammal is a human.
19. A pharmaceutical composition for treating tumor growth and metastasis in a mammal,
said composition comprising a therapeutically effective amount of allogeneic effector
cells together with trifunctional bispecific or trispecific antibodies having the
following properties:
a) binding to a T cell
b) binding to at least one antigen on a tumor cell
c) binding via their Fc portion in the case of trifunctional bispecific antibodies
or via a third specificity in the case of trispecific antibodies to Fc receptor positive
cells;
together with pharmaceutically acceptable carriers and auxiliary substances wherein
the trifunctional antibodies redirect the allogeneic cells away from host tissues
in order to substantially reduce or avoid a graft versus host disease.